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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) Internationa] Patent Classification 6 ;
C12M 1/40, C25B 11700

(11) International Publication Number: WO 95/22597

(43) International Publication Date: 24 August 1995 (24.08.95)

(21) International Application Number: PCT7US95/02157

(22) International Filing Date: 21 February 1995 (21.02.95)

(30) Priority Data:
08/200,174

22 February 1994 (22.02.94) US

(71) Applicant: BOEHRINGER MANNHEIM CORPORATION

[US/US]; 9115 Hague Road, P.O. Box 50528, Indianapolis,
IN 46250 (US).

(72) Inventors: DIEBOLD, Eric, R.; 12580 Ensley Drive, Fishers,

IN 46038 (US). KORDAL, Richard, J.; 11073 Clarkston
Road, 23onsvillc, IN 46077 (US). SURRIDGE, Nigel, A.;
1248 E. 90th Street, Indianapolis, IN 46240 (US). WILSEY,
Christopher, D.; 516 Oak Drive, Carmel, IN 46032 (US).

(74) Agents: YOUNG, D., Michael et al.; Boehringer Mannheim
Corporation, 91 15 Hague Road, P.O. Box 50528, Indianapo-
lis, IN 46250 (US).

(81) Designated States: CA, JP, MX, European patent (AT, BE,
CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT,
SB).

Published

With international search report.

(54) Title: METHOD OF MAKING SENSOR ELECTRODES

=9-2

Ntmmmtm

(57) Abstract

A method for fabricating high-resolution, biocompatible electrodes (1 1) is disclosed, allowing production of an electrochemical sensor
which is capable of precise analyte concentration determination on a very small sample size. Electrically conducting material (3) is affixed
to a first insulating substrate (4). A second insulating substrate (5) is then affixed to the electrically conducting material (3) and patterned
using photolithography to define an electrode area (8). Alternatively, the electrically conducting material (3) may be screen printed directly
onto a standard printed circuit board substrate (4) in the case of a counter or reference electrode. In either case, the substrate may be
rigid or flexible. When the electrodes produced in accordance with the present invention are then used in an electrochemical sensor which
includes a reagent, the small and highly-defined electrode areas (8, 9) permit highly-accurate electrochemical analyte measurements to be
performed on very small sample sizes.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Austria

United Kingdom

Mauritania

Australia

Georgia

Malawi

Barbados

Guinea

Niger

Netherlands

Belgium

Greece

Burkina Paso

Hungary

Norway

Bulgaria

Ireland

New Zealand

Benin

Italy

Poland

BiazS

Japan

Portugal

Belarus

Kenya

Romania

Canada

Kyrgystan

Central African Republic

Democratic People's Republic

Sudan

Congo

of Korea

Sweden

Switzerland

Republic of Korea
Kazakhstan

'Slovenia

Cote <Tlvoire

Slovakia

Cameroon

Senegal

China

Sri Lanka

Chad

Czechoslovakia

Togo

Czech Republic

Latvia

Tajikistan

Germany

Monaco

Trinidad and Tobago

Denmark

Republic of Moldova

Ukraine

Spain

Madagascar

United States of America

Finland

Mafi

Uzbekistan

Fiance

Mongolia

Viet Nam

Gabon

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METHOD OF MAKING SENSOR ELECTRODES

FIELD OF THE INVENTION

5 This invention relates to electrochemical sensors and to a process for fabricating

electrodes for use in electrochemical sensors.

BACKGROUND OF THE INVENTION

The use of sensors in the medical field for testing various blood analytes and in the

10 environmental field for monitoring water or soil contamination is well known. Many of these
sensors perform an electrochemical measurement by applying a potential difference across
two or more electrodes which are in contact with a reagent and sample. Two-electrode
sensors are known which include a working electrode and either a counter or a
reference/counter ("reference") electrode. Three-electrode sensors are also known which

1 5 have a working electrode, a counter electrode, and a reference electrode. Since the area of
the working electrode in any of the above sensor designs has a direct effect on the amount of
current measured, it is highly desirable to fabricate sensors which have a precisely-defined
working electrode area.

Fabricating electrodes for use in sensors has been accomplished by cutting and

20 sealing, "thick-film" or "screen printing", and "thin-film" deposition methods (commonly
used in the production of integrated circuits). Recently, photolithography has also been used
to pattern electrodes on the surface of a substrate. While some of these techniques permit
precise electrode sizing and placement on the support substrate, the ability of sensors made
from such electrodes to make precise measurements is limited by the definition of the

25 working electrode area.

Printed circuit boards ("PCBs") and flex circuits are widely used in the electronics
industry as a means of interconnecting electrical components. There are two basic systems
used to produce PCBs and flex circuits. One is called the "additive method" and the other is

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called the "subtractive method". With the additive method, the desired circuit pattern is built
on top of a non-conductive plastic, ceramic, or other substrate. In the subtractive method, a
non-conductive substrate (e.g., epoxy bonded fiberglass in the case of PCBs, polyiraide in the
case of flex-circuits) is laminated with a copper foil. The copper is then patterned using
5 standard photolithography and wet chemical etching techniques. The copper circuit may
subsequently be plated with nickel, gold, or other metal.

The metal patterning techniques described above which are common to the PCB
industry, however, are unsuitable for biological applications (e.g., analyte sensing). The
plating of metal onto a copper-clad substrate, as described above, results in an iiTegular,
1 0 granular surface that allows penetration of a biological fluid to the underlying copper, thus
giving rise to background electrochemical signals that interfere with measurements. In
addition, copper and nickel are themselves electroactive at the potentials commonly used for
sensing, and therefore cannot be used as a working electrode.

15 SUMMARY O F THE INVENTION

This invention is based on the novel adaptation of some techniques common to the
PCB industry to produce high-resolution electrodes for use in an electrochemical sensor. The
electrodes produced in accordance with the present invention have highly defined and
reproducible size and shape, and importantly have a precisely-defined working electrode

20 area. When the electrodes are then used in an electrochemical sensor, highly-accurate

electrochemical measurements may be performed on veiy small sample sizes. A significant
advantage to the present invention (when the sensor is used to detect or measure an analyte in
a blood sample) is the low blood sample volume required for the electrochemical
measurement, thus allowing for a very low pain lancet device which produces low sample

25 volumes. Since in one embodiment the electrodes are manufactured on separate pieces of
substrate material, another advantage of the present invention is the separation of the
fabrication processes of the two electrodes, which allows separation of the chemistries
associated with the working and the counter electrodes.

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Fabricating an electrode in accordance with the present invention involves first
attaching a high quality thin metal film (rather than copper foil laminates) to a bare rigid or
flexible substrate. A layer of photoresist is then applied to the thin metal layer and patterned
using photolithography to precisely define an electrode area and a contact pad. Importantly,
5 the photoresist layer is not removed after patterning and acts as an insulator in the finished
electrochemical sensor. Alternatively, a dielectric material may be screen printed directly to
the metal layer in a pattern which defines the electrode area and contact pad. In the case of a
reference or counter electrode, the metal may be applied directly to a standard PCB substrate.
The electrodes described above may then be used to fabricate a novel
1 0 electrochemical sensor in which the electrodes are arranged either in opposing or adjacent
form. When a reagent is applied to one or both exposed electrode areas, an electrochemical
detection and/or measurement of an analyte in a sample may be performed.

BRIEF DESCRIPTION OF THE PRAWTNCS

15 FIG. 1 shows a method of fabricating a working, counter, or reference electrode

element in accordance with the present invention.

FIG. 2 shows another embodiment of a method of fabricating a working, counter, or
reference electrode element in accordance with the present invention.

FIG. 3 shows a method of fabricating a reference or counter electrode element in
20 accordance with the present invention.

FIG. 4 shows another embodiment of a method of fabricating a reference or counter
electrode element in accordance with the present invention.

FIG. 5 shows an exploded view of the opposing electrode electrochemical sensor in
accordance with the present invention.
25 FIG. 6 shows an assembled view of the opposing electrode electrochemical sensor of

FIG. 5.

FIGS. 7a-7h show a method of fabricating adjacent electrode elements for use in an
adjacent electrode electrochemical sensor in accordance with the present invention.

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FIG. 8a shows a top view of FIG. 7g and FIG. 8b shows a top view of FIG. 7h.
FIG. 9 shows a dose response of one embodiment of a electrochemical sensor in
accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION
5 The adaptation of some PCB fabrication techniques to make electrodes functional in

biological fluids relies on electrochemical inertness in the potential range of interest for
sensing, approximately -1 to +1 volts versus silver/silver chloride (Ag/AgCl). In accordance
with the present invention, high quality thin noble metal films are used as electrodes rather
than copper foil laminates. These thin metal films can be sputtered or evaporatively

1 0 deposited onto an appropriate foil material (e.g., polyester, polycarbonate, polyirnide) and
then laminated to a support substrate (e.g. by Courtaulds Performance Films, Canoga Park,
California). Alternatively, the thin metal films may be deposited directly onto the support
substrate. The resulting metallized substrate displays extremely small and uniform grain size
(10-50 nm (nanometers) diameter), and importantly does not contain copper or other

15 electrochemicalry active materials. Such surfaces are nearly ideal for the purpose of making
electrochemical measurements in biological or corrosive solutions. A second insulating
substrate is then applied to the metal layer and precisely patterned to form an open electrode
area and a meter contact pad. The combination of first insulating substrate, metal, and
second insulating substrate is referred to herein as an "electrode element. 1 *

20 Two types of electrode elements are described below. The "opposing 1 * electrode

element is designed to be used in combination with a second opposing electrode element,
separated by a spacer in a "sandwich** fashion. This embodiment is referred to as the
"opposing electrode electrochemical sensor." The opposing electrode electrochemical sensor
includes a working electrode element and either a counter or a reference electrode element as

25 described below. The "adjacent" electrode elements are fabricated on the same substrate
side-by-side in a parallel fashion. This embodiment is referred to as the "adjacent electrode
electrochemical sensor." The adjacent electrode electrochemical sensor may include a

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working electrode element and either a counter or a reference electrode element, or may
include a working, counter and reference electrode element

FABRICATION OF OPPOSING ELECTRODE ELEMENTS FOR THE OPPOSING

5 ELECTRODE ELECTROCHEMICAL SENSOR

A working, counter, or reference electrode element may be produced in accordance
with the present invention as shown in FIG. 1 . Electrically conducting material 1 (e.g., a
noble metal or carbon) is vacuum sputtered or evaporatrvery deposited onto thin support
material 2 (e.g., polyimide or other polymer such as polyester, polyethylene terephthalate

1 0 (PET), or polycarbonate) to form metallized thin support material 3 (e.g., by Courtaulds
Performance Films, Canoga Park, California). This step may or may not be preceded by
depositing, with the same means, a thin anchor layer of chromium, titanium, or other suitable
material (not shown in FIG. 1 ). The purpose of the thin anchor layer is to increase adhesion
between electrically conducting material 1 and thin support material 2, as well as to stabilize

15 the microstructure of electrically conducting material 1 .

Alternatively, electrically conducting material 1 can be deposited onto the surface of
thin support material 2 by the method of electroless plating or a combination of activation
and electroplating. These processes are well known but will be briefly described. With
electroless plating, thin support material 2 is cleaned and if necessary subjected to a surface

20 roughening step. The surface of thin support material 2 is men chemically treated or

"activated" with a colloidal catalyst (e.g., PdCfe-SnCk hydrosol) mat adsorbs strongly onto
the surface. The substrate and adsorbed catalyst should then be treated in an "accelerator
bath", as is commonly known in the electroless plating art, using an acidic bath containing
PdCl 2 . Finally, thin support material 2 is plated in an electroless plating bath designed to

25 deposit a thin layer of electrically conducting material 1 onto the surface of thin support
material 2.

With electroplating, thin support material 2 is first activated using a commercial
surface treatment (such as that available from Solution Technology Systems, Inc.). Thin

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support material 2 may then be electroplated in a manner well known to the electroplating
industry with electrically conducting material 1, thereby forming metallized thin support
substrate 3.

Metallized thin support material 3 is then laminated (e.g., by Litchfield Precision
5 Components, Litchfield, Minnesota) to first insulating substrate 4 (e.g., a bare fiberglass
circuit board such as 10 mil thick FR4 from Norplex/Oak, La Crosse, Wisconsin, available as
product ED 130) using a suitable laminating adhesive system (e.g., Z^FLEXTM adhesive
system from Courtaulds Performance Films, Canoga Park, California). First insulating
substrate 4 could be any suitable non-conductive glass or plastic substrate with the desired

1 0 supportive rigidity. In this step metallized thin support material 3 and first insulating
substrate 4 could optionally be laminated using a hot press.

Once metallized thin support material 3 is supported on first insulating substrate 4,
metallized thin support material 3 can be processed with a suitable solder resist to form an
electrode area and a contact pad area for insertion into a meter and a power source. The

1 5 surface of metallized thin support material 3 is cleaned with a suitable solvent system (e.g., a
chloroflurocarbon solvent) and coated with second insulating substrate 5, a commercial
solder resist, either by screen printing or flood coating and then dried according to the
manufacturer's specifications. An example of a commercial solder resist that could be used is
ENPLATE®DSR-3242 solder resist from Enthone-OMI, Inc. (a negative resist). The second

20 insulating substrate 5 is exposed to uhra-violet light rays 7 through photomask 6. As a result,
a latent image is generated in second insulating substrate 5 rendering it insoluble in a
developer solution in those areas that were exposed to uhra-violet rays 7. Before developing,
mask 6 is removed. Hie type of developer solution that should be used is process-dependent
and generally will be specified by the manufacturer of the resist Processing in the developer

25 solution removes portions of second insulating substrate 5, thus forming first cutout portion 8
and second cutout portion 9. Following this procedure, the remaining second insulating
substrate 5 may be permanently cured by a suitable combination of heat and ultra-violet light,
making it a good barrier layer for applications in biological fluids. In addition to the negative

WO 95/22597 PCT/US95/02157

solder resist described above, positive resists may also be used in accordance with the present
invention. In the case of a positive solder resist, the resist used is insoluble in the developing
solution, unless the resist is exposed to electromagnetic radiation as specified by the
manufacturer of the resist.
5 As a result of the photolithographic process described above, first cutout portion 8

and second cutout portion 9 are formed in second insulating substrate 5, exposing the
underlying metallized thin support material 3. In finished electrode element 1 1, the area of
first cutout portion 8 defines the electrode area and second cutout portion 9 acts as a contact
pad between electrode element 1 1 and a meter and a power source. When electrode element
10 1 1 is a reference electrode element, a reference electrode material (e.g., #DB2268

silver/silver chloride ink from Acheson Colloids Co., Port Huron, Michigan) is additionally

applied to the electrode area defined by first cutout portion 8.

Importantly, although it is common when using photolithography to remove the resist
layer, in the present invention second insulating substrate 5 is not removed and acts as an

15 insulating substrate in the finished electrochemical sensor. In addition, vent port 10, which
extends through second insulating substrate 5, metallized thin support material 3, and first
insulating substrate 4, may be included and used as a vent port for the capillary space
(described below) in the finished electrochemical sensor and/or as a means of introducing the
sample to the capillary space. At this stage, any reagent that is required may be dispensed

20 onto the appropriate electrode areas as described below.

As an alternative to applying the second insulating substrate and performing
photolithography to define the working electrode area and contact pad as described above, a
thin-film dielectric material may be screen printed onto metallized thin support material 3.
The thin-film dielectric material may be UV-curable (e.g., #ML-25 1 98 from Acheson

25 Colloids or #501 8 from DuPont Electronics) or heat-curable (e.g., #7192M from Metech).
The thin-film dielectric material can be applied through a screen in a specific pattern so as to
define first cutout portion 8 and second cutout portion 9 in the thin-film dielectric material,
exposing the underlying metallized thin support material 3. In the finished electrode element,

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the area of first cutout portion 8 defines the electrode area and second cutout portion 9 acts as
a contact pad between the electrode element and a meter and a power source. The thin-film
dielectric material can be chosen such that it may be cross-linked photochemically after
application to the metallized thin support material, thus increasing stability and adhesion to
5 the surface of the metallized thin support material as well as forming an impenetrable barrier
layer for use in biological media. The thin-film dielectric material also acts as an insulating
substrate in the finished electrochemical sensor. A vent port may also be included and used
as a means of introducing the sample in the finished electrochemical sensor as discussed
above.

1 0 Another method that may be used to fabricate a working, counter, or reference

electrode element in accordance with the present invention is shown in FIG. 2. In this
embodiment, the electrically conducting material is deposited directly onto a more flexible
first insulating substrate, thus facilitating a less-expensive, semi-continuous production
method. Electrically conducting material 12 is vacuum Sputtered or evaporati very deposited

15 directly onto first insulating substrate 13 (e.g., by Courtaulds Performance Films, Canoga
Park, California). An example of a suitable substrate is MYLAR™ substrate (from DuPont)
of approximately 10 mil thickness. Other suitable plastic, glass or fiberglass substrates may
also be used. Alternatively, electroless or electroplating techniques as described above could
be used to deposit metal 12 onto first insulating substrate 13.

20 Electrically conducting material 12 is then coated with second insulating substrate

14, such as a liquid negative solder resist (e.g., PROBOMER™ solder resist from Ciba-
Geigy) via a flood or dip coating while still in a roll form and then dried using a suitable
combination of infrared and thermal heating. Second insulating substrate 14 is exposed to
ultra-violet light rays 16 through photomask 15. A latent image is generated in second

25 insulating substrate 14 as described above and following removal of mask 15 and processing
in the developer solution, portions of second insulating substrate 14 are removed forming
first cutout portion 17 and second cutout portion 1 8. (As an alternative to the application of
second insulating substrate 14, it is also possible to screen print a layer of dielectric ink in a

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specific pattern equivalent to that obtained via the exposure process disclosed above.)
Second insulating substrate 14 may also be permanently cured as described above. In
addition, solder resists other than described above (e.g., positive resists) may be used in
accordance with the present invention.
5 In finished electrode element 20, the area of first cutout portion 1 7 defines the

electrode area and second cutout portion 1 8 acts as a contact pad between electrode element
20 and a meter and a power source. As described above, when electrode element 20 is a
reference electrode element, a reference electrode material (e.g., #DB2268 silver/silver
chloride ink from Acheson Colloids Co., Port Huron, Michigan) is additionally applied to the

1 0 electrode area defined by first cutout portion 1 7.

The method described above for producing electrode elements utilizing a flexible
first insulating substrate allows for a continuous production process, in which the metal is
deposited on a roll of the first insulating substrate. The metallized plastic roll is then coated
with the second insulating substrate and processed through an in-line exposure tool to expose

15 a series of the desired patterns (electrode areas and contact pads) in the second insulating
substrate along the roll. This is followed by a developing cycle, according to the
manufacturer's specifications and familiar to those skilled in the art, followed by a curing
cycle. This results in similarly exposed areas of metal for the electrode areas and the contact
pad areas, although the array of multiple electrodes is supported on a continuous roll of

20 plastic. Reagent is then dispensed onto the electrode areas defined in the second insulating
substrate. An adhesive spacer layer (described below) is then applied via continuous roll
lamination to the second insulating substrate (or dielectric ink). A second roll of electrodes is
then fabricated as described above and laminated to the first roll so as to form a capillary
chamber which exposes the active electrode areas as well as the reagent The multiple

25 sensors so defined on a continuous roll of material are then punched or die cut from the web
prior to packaging.

As described above, a standard PCB substrate (a copper layer laminated to a
fiberglass substrate) is inappropriate for use as a working electrode in an electrochemical

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sensor since it interferes with the electrochemical measurement. Specifically, when a
mediator is being oxidized at the working electrode surface (anodic process), copper may
also be oxidized and therefore interfere with the electrochemical measurement. However,
when reduction is occurring at the surface of a reference or counter electrode (cathodic
5 process), a standard PCB substrate may be used in the reference or counter electrode since
copper will not be reduced and therefore will not interfere. One embodiment of a reference
or counter electrode using a standard PCB as the first insulating substrate will now be
described.

Referring to FIG. 3, a standard PCB substrate, which includes copper layer 30

1 0 laminated to fiberglass substrate 3 1, is used as a first insulating substrate. Electrically

conducting material 32 (e.g., #DB2268 silver/silver chloride ink from Acheson Colloids, Port
Huron, Michigan) may be screen printed directly onto copper layer 30, leaving cutout portion
33 exposed. Finally, spacer 34 (e.g., MYLAR™ substrate with double-sided adhesive),
which includes first cutout portion 35 and second cutout portion 33, is placed on top of

1 5 electrically conducting material 32. Spacer 34 may also be any other suitable plastic or
fiberglass. First cutout portion 35 and second cutout portion 33 may be cut out by using a
laser process (e.g., by Laser Machining Inc., Somerset, Wisconsin). In finished reference or
counter electrode element 37, the area of first cutout portion 35 exposes underlying
electrically conducting material 32 and defines the reference or counter electrode area.

20 Second cutout portion 33 exposes underlying copper layer 30 and acts as a contact pad
between reference or counter electrode element 37 and a meter and a power source. In
addition, vent port 36, which extends through spacer 34, electrically conducting material 32,
copper layer 30, and fiberglass substrate 31, may be included and used as a vent port for the
capillary space and/or as a means of introducing the sample to the capillary space as

25 described above.

Another method that may be used to fabricate a reference or counter electrode
element in accordance with the present invention is shown in FIG. 4. A thin anchor or
stabilizing layer of first electrically conducting material 38 (e.g., palladium) is sputtered or

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evaporatively deposited onto thin support material 40, followed by a thicker layer of second
electrically conducting material 39 (e.g., silver), to form metallized thin support material 41
(e.g., by Courtaulds Performance Films, Canoga Park, California). As described above, thin
support material 40 may be a polyimide or other polymer such as polyester, PET, or
5 polycarbonate. Metallized thin support material 4 1 may then be laminated to first insulating
substrate 42, which may be fiberglass, glass, or plastic as described above. Alternatively,
first electrically conducting material 38 may be directly sputtered or evaporatively deposited
onto first insulating substrate 42 rather than onto thin support material 40. Spacer 43, which
includes first cutout portion 44 and second cutout portion 45, is placed on top of metallized

1 0 thin support material 41 . Spacer 43 may be MYLARTM substrate with double-sided adhesive
as described above or any other suitable plastic or fiberglass. Finally, when second
electrically conducting material 39 is silver, a solution of FeCb (not shown) may be
dispensed into first cutout portion 44 of spacer 43, where a layer of silver chloride 46 is
formed by an oxidative process. The process of defining a reference electrode area can also

1 5 optionally be assisted by applying and patterning a photoresist layer onto the surface of
metallized thin support material 41 prior to treatment with FeCfe. Alternatively, selected
regions of metallized thin support material 41 may be dipped into solutions of FeC^ to
achieve the same result In finished reference or counter electrode element 48, the area of
first cutout portion 44 exposes layer 46 and defines the reference or counter electrode area.

20 Second cutout portion 45 exposes metallized thin support material 41 and acts as a contact

..." *•■..«■_...■

pad between reference or counter electrode element 48 and a meter and a power source. In
addition, vent port 47, which extends through spacer 43, metallized thin support material 41,
and first insulating substrate 42, may be included and used as a means of introducing the
sample in the finished electrochemical sensor as described above.

OPPOSING ELECTRODE ELECTROCHEMICAL SENSOR
One embodiment of an electrochemical sensor with an opposing electrode design in
accordance with the present invention is shown in FIGS. 5 and 6. Reference or counter

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electrode element 48 is spatially displaced from working electrode element 1 1 by spacer 43.
(Spacer 43 is normally affixed to reference or counter electrode element 48 during
fabrication, but has been shown separate from element 48 for the purpose of FIG. 5.) First
cutout portion 44 in spacer 43 forms capillary space 49 when situated between reference or
5 counter electrode element 48 and working electrode element 1 1 . First cutout portion 8 in
working electrode element 1 1 exposes metallized thin support material 3, the working
electrode area, which is exposed to the capillary space 49. First cutout portion 44 in spacer
43, when affixed to reference or counter electrode element 48, defines reference or counter
electrode area 46 (shown in phantom lines in FIG. 5), which is also exposed to capillary
10 space 49. Second cutout portions 9 and 45 expose metallized thin support materials 3 and 41
respectively and act as contact pads between electrochemical sensor 52 and a meter and a
power source.

In assembled electrochemical sensor 52 shown in FIG. 6, capillary space 49 (shown
in phantom lines) has first opening 50 at one edge of the electrochemical sensor. In addition,

15 vent port 10 in working electrode element and/or vent port 47 in reference or counter

electrode element 48 may be used to provide second opening 51 into capillary space 49. The
vent port may optionally be used as a means of introducing the sample to the capillary space.
In use, a sample containing an analyte to be detected or measured may be introduced into
capillary space 49 of electrochemical sensor 52 through either opening 50 or vent port 5 1 . In

20 either case, the sample is spontaneously drawn into the electrochemical sensor by capillary
action. (Preferably, a surfactant is included in the capillary space to aid in drawing the
sample into the capillary space.) As a result, the electrochemical sensor automatically
controls the sample volume measured without user intervention. In addition, since the
sample is totally contained within capillary space 49, contamination of the meter into which

25 electrochemical sensor 52 is inserted and the patient could be reduced or eliminated, a
significant advantage when the sample is blood or a biological fluid.

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FABRICATION OF ADJACENT ELECTRODE ELEMENTS FOR THE
ADJACENT ELECTRODE ELECTROCHEMICAL SENSOR
Adjacent electrode elements may also be produced in accordance with the present
invention to form an adjacent electrode electrochemical sensor as indicated in FIGS. 7 & 8.
5 The process is similar to that described above for the opposing electrode elements. However,
since the electrodes are on the same support substrate next to each other, an additional metal
etching step is involved. Electrically conducting material 61 (e.g., a noble metal) is vacuum
sputtered or evaporatively deposited onto thin support material 62 (e.g., poryimide or other
polymer such as polyester, PET, or polycarbonate) to form metallized thin support material

10 63 as described above. (FIGS. 7a-7b.) This step may or may not be preceded by depositing a
thin anchor layer. Alternatively, electrically conducting material 61 can be deposited onto
the surface of thin support material 62 by the method of electroless plating or a combination
of activation and electroplating as described above. Metallized thin support material 63 is
then laminated to first insulating substrate 64 (e.g., a bare fiberglass circuit board such as 10

1 5 mil thick FR4) using a suitable laminating adhesive system (e.g., Z-FLEXTM adhesive system
from Courtaulds Performance Films, Canoga Park, California). (FIG. 7b.) First insulating
substrate 64 could be any suitable non-conductive glass or plastic substrate as described
above. In this step metallized thin support material 63 and first insulating substrate 64 could
also be laminated using a hot press.

20 The surface of metallized thin support material 63 is then cleaned with a suitable

solvent system and then coated with photoactive etch resist 65. (FIG. 7c.) Either positive or
negative etch resists may be used. The coating method will depend on whether a semi-
aqueous or liquid resist is used. The serai-aqueous resists are generally applied by a
lamination process, whereas the liquid resists are dip-coated, spray-coated, curtain-coated, or

25 screen printed. Specifically, in the case of a negative, semi-aqueous resist from DuPont, sold
under the mark RESISTON, the resist is applied by a hot roll lamination process.
Photoactive etch resist 65, metallized thin support material 63, and first insulating substrate
64 are then exposed to ultra-violet light 67 through photomask 66 and baked for 15 minutes

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at 180°F. (FIG. 7d.) As a result, a latent image is generated in photoactive etch resist 65
rendering it insoluble in a developer solution in those areas that were exposed to ultra-violet
rays 67. Processing in the developer solution removes the unexposed areas of photoactive
etch resist 65, thus exposing portions of underlying metallized thin support material 63.
5 (FIG. 7e.)

The entire substrate is then placed in a bath containing a chemical etchant (e.g , when
electrically conducting material 61 is gold, an aqua regia or a solution of KI and may be
used) and incubated with constant stirring at a controlled temperature. The etchant dissolves
the exposed metallized thin support material 63, but is unable to dissolve the portions of

1 0 metallized thin support material 63 that are covered with photoactive etch resist 65. (FIG.
7f ) Photoactive etch resist 65 is then removed with a solvent revealing metallized thin
support material 63 in the desired electrode pattern. (FIGS, 7g & 8a.) The electrode pattern
may include, for example, contact pads 69, leads 70, and electrode areas 71. (FIG. 8a)
Finally, leads 70 are insulated with second insulating substrate 68, which may be a solder

1 5 resist or a screen printable dielectric as described above for the opposing electrode design.
(HCS.7h&8b.)

In accordance with the present invention, the counter electrode may then optionally
be converted to a reference electrode by electroplating silver directly onto the counter
electrode, followed by treatment with FeCl 3 to convert the silver surface to silver chloride.

20 To facilitate this process a sacrificial interconnecting bus could be designed into the layout to
allow multiple electrodes to be electroplated in one step. The other areas of metal would
need to be protected during the plating step since it is generally done as a batch process. This
could be accomplished with an etch resist in a maimer similar to that described above for the
adjacent working/counter electrode arrangement Alternatively, a layer of reference electrode

25 material (e.g., silver chloride ink) may be screen printed on top of the metal layer to yield a
reference electrode.

WO 95/22597

PCT/US95/02157

REAGENT

Many different types reagents may be applied to the working electrode and/or the
reference or counter electrode to provide for a fully functional sensor whose signal is
selective for and sensitive to the concentration of an analyte (e.g., glucose). These reagents
5 can be dispensed onto the working electrode area of the electrochemical sensors described
above using an automated mechanical dispenser, screen printing, slot or roll coating, spin
coating, blade coating, or ink-jet printing. (Sometimes, bom working and counter electrode
areas will be coated with a reagent) The reagents thus dispensed form a thin coating over the
electrode which is rapidly swollen upon application of the sample (e.g., blood), at which time
10 a suitable potential may be applied to the electrodes and a current measurement made. The
current measurement may then be related to the concentration of the target analyte in the
sample. The use of polymeric materials and a capillary chamber to contain the reagent
greatly reduces the risk of contamination by chemicals in the sensor of the open wound in the
patient's finger.

15 An example of a reagent that may be used with the present invention for the detection

of glucose in a whole blood sample, designed to be used with the opposing electrode
electrochemical sensor having a working electrode element and a reference electrode
element, will now be described. The components of the reagent are listed below in table 1 .
20 Table 1 - reagent components

Component

Amount

2-(N-morpholino) cthancsulphonic acid
(MES Buffer)

100nuUimolar(mM)

Triton X-100

0.08%wtAvt

Polyvinyl alcohol (PVAX raol; weight 1 OK,
88% hydrolyzed

1.00%wt/wt

Imidazole osmium mediator (reduced form -
synthesis described below)

62mM

Glucose Oxidase

6000 raiits/ml !

Following is a description of how the reduced form of the imidazole osmium
mediator was synthesized. The osmium intermediate (Os^py^C^) was first synthesized,
followed by the reduced form of the imidazole osmium mediator [Os(IIXbpyi(im)Cir H [CI]".

WO 95/22597 PCTAJS95/02157

("bpy" is a shorthand abbreviation for 2-2-bipyridine and "im" is a shorthand abbreviation
for imidazole.)

SYNTHESIS OF OSMIUM INTERMEDIATE

1) 19335 g K 2 0sCl 6 (0.04019 mole - from Aldrich) and 13.295 g bpy (0.08512 mole
5 - from Aldrich) were weighed and transferred into a 1000 ml 1-neck flask.

2) 400 ml N,N'-dimethylfonnamide (DMF - from Mallinckrodt) was added to the
flask to dissolve all reactants.

3) The flask contents were heated to reflux (152-54°C) with stirring. Reflux was
maintained for 1 hour with lower heat (setting was decreased from 100% to 65% on variable

1 0 transformer) to avoid overboiling.

4) The heat was turned off and the flask was cooled with continued stirring to 30-
40°Cin 1-2 hours.

5) The mixture was filtered with vacuum using a medium grade glass fritted filter
(150 ml).

1 5 6) The flask was rinsed with 20 ml DMF and poured into the filter.

7) The filtered DMF solution was transferred to a 3 liter (1) beaker.

8) 22.799 grams Na2S20 4 (from Mallinckrodt) was weighed and transferred to a
separate 2 1 beaker.

9) 2 I deionized water was added to the beaker to dissolve the NqgS^*

20 10) The Na 2 S20 4 aqueous solution was transferred to a dropping funnel and added

dropwise (about 5 drops/second), over a period of 45 minutes, to the stirring DMF solution.

1 1) The mixture was cooled in an ice bath for more than 3 hours.

12) The cooled mixture was filtered with vacuum using Whatman qualitative filter
paper in a ceramic filter.

25 13) The filtered product was washed twice with 50 ml H 2 0; twice with 50 ml

methanol; and twice with 50 ml diethyl ether.

14) The product, OsfrpykCk, was dried under high vacuum (about 30 in. Hg) at
50°C for more than 15 hours (overnight).

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15) The product was weighed, transferred to a brown bottle having a screw-on cap,
and stored in desiccator at room temperature. Yield: theoretical = 23 .3 5 g, actual = 1 5.56 g,
yield = 66.6%.

SYNTHESIS OF THE REDUCED FORM OF THE IMIDAZOLE OSMIUM
5 MEDIATOR

1) 14.01 g OsCbpy^Cb (0.0244 mole) and 2.30 g imidazole (0.0338 mole - from
Aldrich) were weighed and transferred into a 2000 ml 1-neck flask.

2) 600 ml ethanol and 600 ml deionized water were added to dissolve all reactants.

3) The flask contents were heated to reflux with stirring and reflux was maintained
10 for 6 hours with lower heat (setting was decreased from 90% to 60% on variable transformer)

to avoid overboiling.

4) The heat was turned off and the flask cooled with continued stirring to 30-40°C
over a period of 1 hour.

5) Half of the solution was transferred to a 1000 ml 1-neck flask and the solvents
1 5 were rotary evaporated. The remainder of the solution was added to the flask and the

solvents were rotary evaporated.

6) The dried product was rinsed on the flask wall with 50 ml ether and the ether
wash was discarded.

7) The product was dried under high vacuum (about 30 in. Hg) at 50°C for more than
20 15 hours (overnight).

8) The flask wall was scraped to collect the product, [Os(II)(bpy^(im)Cl] + [Cl]".
The product was weighed and transferred to a brown bottle having a screw-on cap. The
bottle was stored in a desiccator at room temperature. Yield: theoretical = 163 g, actual =
16.1 g, yield = 98.8%.

25 Following is a description of how the reagent described in table 1 was prepared and

used in combination with opposing electrode elements to form an electrochemical sensor.
1) Polymer matrix

WO 95/22597 PCT/US95/02157

a) 1 .952 g MES buffer was added to 85 ml nanograde water. The mixture
was stirred until dissolved. The pH of the solution was adjusted to 5.5
with NaOH and the total volume of the solution was brought to 100 ml.

b) 0.08 g of Triton X-100 and 1 g of PVA was added to a 150 ml beaker,

5 Buffer solution was added to bring the total weight of the solution to 1 00

g. The mixture was then heated to boiling to dissolve the PVA.
2) Coating mixture

a) 4.0 mg of the reduced osmium mediator, [Os^Xbpy^im^J+lCl]", was
added to 1 ml of the polymer matrix. The mixture was vortexed to
10 dissolve the mediator. 6000 units of glucose oxidase was added to the

mixture and the solution was mixed until the enzyme was dissolved.
Although the reagent described above is preferred for use with this invention, other
types of reagents, which are specifically reactive with an anaryte in a fluid sample to produce
an electrochemically-measurable signal which can be correlated to the concentration of the
1 5 anaryte in the fluid sample, may be used. The reagent should include at least a mediator and
an enzyme. Preferably, the reagent should also includes a buffer, a film former, and a
surfactant as described above.

Other redox mediator systems could also be utilized (e.g., using potassium
ferricyanide as the redox mediator rather than the imidazole osmium mediator described
20 above) as well as redox polymer systems (in which the mediator and enzyme are immobilized
on the electrode surface).

USE OF THE ELECTROCHEMICAL SENSOR
The electrochemical sensor described above may be used for, but is not limited to,
25 the determination of blood glucose levels using a small drop of blood (3-20ul) obtained from
the patient's finger or other location by the use of a lancing device. A significant advantage
to the present invention is the low volume required for the measurement, thus allowing for a
very low pain lancet device which produces low sample volumes.

WO 95/22597

PCT/US95/02157

An example of how an opposing electrode electrochemical sensor was made and used
to determine the concentration of glucose in a whole blood sample will now be described. A
reference electrode element was fabricated as described above, having gold as the electrically
conducting material and having a spacer attached to expose a portion of the gold (capillary
5 space). A silver/silver chloride polymer thick film ink (Acheson Colloids DB 2286) was
thinned 2:1 wtAvt with butoxyethanol. 2.5 \i\ of the resulting mixture was applied to the
capillary space and spread to fill the capillary area. The electrode was then dried for 15
minutes at 90°C.

A working electrode element was fabricated as described above, having gold as the

10 electrically conducting material. 1 jil of the coating mixture (from the reagent example

described above) was then applied to the working electrode surface of the working electrode
element. The coated electrode was dried at 45°C for 1 5 minutes.

The working electrode element was then "sandwiched" together with the reference
electrode element as described above and as illustrated in FIGS. 5 & 6 to form the completed

15 electrochemical sensor. The completed electrochemical sensor was used, as described below,
to perform a glucose assay. The working electrode potential was made +200 millivolts (mv)
versus the Ag/AgCl reference electrode by a potentiostat 10 jil of spiked giycolyzed venous
blood was added to capillary space 49 through first opening 50. Current was measured 10
seconds after applying the sample to the electrochemical sensor. FIG. 9 shows a dose

20 response curve generated by the assay of spiked giycolyzed venous blood samples with
different levels of glucose.

It is intended that an electrochemical sensor made in accordance with the present
invention should be inserted into a small meter device where the contact tabs can make
electrical contact with the measuring circuit within the meter. The meter will normally be

25 adapted to apply an algorithm to the current measurement, whereby the analyte level is
provided and visually displayed. Examples of improvements in such a power source and
meter are the subject of commonly assigned U.S. Patent Number 4,963,814 - "Regulated
Bifurcated Power Supply" (Parks et al. T issued October 16, 1990), U.S. Patent Number

WO 95/22597 PCTYUS95/02157

4,999,632 - "Analog to Digital Conversion with Noise Reduction" (Earks, issued March 12,
1991), U.S. Patent Number 4,999,582 - "Electrochemical sensor Electrode Excitation Circuit"
(Plfffa gtri,, issued March 12, 1991), and U.S. Patent No. 5,243,516 - "Biosensing
Instrument and Method" (White, issued September 7, 1993), the disclosures of which are
5 hereby incorporated by reference.

The present invention has been disclosed in the above teachings and drawings with
sufficient clarity and conciseness to enable one skilled in the art to make and use the
invention, to know the best mode for carrying out the invention, and to distinguish it from
other inventions and from what is old. Many variations and obvious adaptations of the
1 0 invention will readily come to mind, and these are intended to be contained within the scope
of the invention as claimed below.

WO 95/22597

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CLAMS

What is claimed is:

1 . An electrochemical sensor useful for measuring the concentration of an analyte in
a fluid sample, comprising:
5 (a) opposing working and counter electrode elements, spatially displaced by a spacer

having a first cutout portion forming a capillary space between the working and
counter electrode elements and a second cutout portion allowing electrical
connection between the counter electrode element and a meter and power source,
wherein the capillary space is vented by a port in the working or counter electrode
1 0 elements, the working electrode element including

1) a first insulating substrate,

2) an electrically conducting material affixed to the first insulating

3) a second insulating substrate affixed to the electrically conducting
1 5 material and the spacer, the second insulating substrate having a first

cutout portion for exposing a portion of the electrically conducting
material to the capillary space and a second cutout portion which
allows contact between the electrically conducting material and the
meter and power source,
20 the counter electrode element including

1) an insulating substrate, and

2) an electrically conducting material affixed to the insulating substrate
and the spacer; and

(b) a reagent disposed in the capillary space, the reagent being specifically reactive
25 with the analyte in the fluid sample to produce an electrochemical ry-measurable

signal which can be correlated to the concentration of the analyte in the fluid
sample.

WO 95/22597 PCT/US95/02157

2. The electrochemical sensor of claim 1, wherein the vent port extends through the
working electrode element and the counter electrode element.

3. The electrochemical sensor of claim 2, wherein the counter electrode element
further comprises:

5 3) a second insulating substrate affixed to the electrically conducting

materia] and the spacer, the second insulating substrate having a first
cutout portion for exposing a portion of the electrically conducting
material to the capillary space and a second cutout portion which
overlays the second cutout portion of the spacer.
10 4. The electrochemical sensor of claim 3, wherein the electrically conducting

material of the working electrode element is a noble metal or carbon.

5. The electrochemical sensor of claim 4, wherein the electrically conducting
material of the counter electrode element is a noble metal or carbon.

6. The electrochemical sensor of claim 5, wherein the second insulating substrate is
15 a solder resist

7. The electrochemical sensor of claim 6, further comprising:

(d) the power source in electrical connection with the working and counter electrode
elements; and

(e) the meter in electrical connection with the working and counter electrode
20 elements and capable of measuring current

8. The electrochemical sensor of claim 7, wherein the reagent comprises a mediator
and an enzyme.

9. The electrochemical sensor of claim 8, wherein the reagent further comprises a
buffer, a film former, and a surfactant

25 1 0. The electrochemical sensor of claim 8, wherein the mediator is

[Os(IIXbpy) 2 (im)Cl] + [Cl]" and the enzyme is glucose oxidase.

WO 95/22597

PCT/US95/02157

1 1 . The electrochemical sensor of claim 9, wherein the analyte is glucose, the
mediator is [Osfll^py^im^IffCir, the enzyme is glucose oxidase, the buffer is MES
buffer, the film former is polyvinyl alcohol, and the surfactant is a nonionic surfactant

12. An electrochemical sensor useful for measuring the concentration of an analyte
5 in a fluid sample, comprising:

(a) opposing working and reference electrode elements, spatially displaced by a

spacer having a first cutout portion forming a capillary space between the working
and reference electrode elements and a second cutout portion allowing electrical
connection between the reference electrode element and a meter and power
1 0 source, wherein the capillary space is vented by a port in the working or reference

electrode elements, the working electrode element including

1) a first insulating substrate, v

2) an electrically conducting material affixed to the first insulating
substrate, and

15 3) a second insulating substrate affixed to the electrically conducting

material and the spacer, the second insulating substrate having a first
cutout portion for exposing a portion of the electrically conducting
material to the capillary space and a second cutout portion which
allows contact between the electrically conducting material and the

20 meter and power source,

the reference electrode element including

1) an insulating substrate,

2) an electrically conducting reference material affixed to the insulating
substrate and the spacer; and

25 (b) a reagent disposed in the capillary space, the reagent being specifically reactive

with the analyte in the fluid sample to produce an electrochemically-measurable
signal which can be correlated to the concentration of the analyte in the fluid
sample.

WO 95/22597

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13. The electrochemical sensor of claim 12, wherein the vent port extends through
the working electrode element and the reference electrode element

14. The electrochemical sensor of claim 13, wherein the reference electrode element
further comprises:

5 4) a second insulating substrate affixed to the reference electrode

material and the spacer, the second insulating substrate having a first
cutout portion for exposing a portion of the electrically conducting
material to the capillary space and a second cutout portion which
overlays the second cutout portion of the spacer.
10 15. The electrochemical sensor of claim 14, wherein the electrically conducting

material of the working electrode element is a noble metal or carbon.

1 6. The electrochemical sensor of claim 1 5, wherein the electrically conducting
reference material of the reference electrode element is silver/silver chloride.

17. The electrochemical sensor of claim 16, wherein the second insulating substrate
15 is a solder resist.

18. The electrochemical sensor of claim 17, further comprising:

(d) the power source in electrical connection with the working and reference
electrode elements; and

(e) the meter in electrical connection with the working and reference electrode
20 elements and capable of measuring current

19. The electrochemical sensor of claim 18, wherein the reagent comprises a redox
mediator and an enzyme.

20. The electrochemical sensor of claim 19, wherein the reagent further comprises a
buffer, a film former, and a surfactant.

25 21. The electrochemical sensor of claim 1 9, wherein the redox mediator is

[Os(n)(bpy)2(im)Crj* [CI]" and the enzyme is glucose oxidase.

WO 95/22597

PCT/DS95/02157

22. The electrochemical sensor of claim 20, wherein the analyte is glucose, the redox
mediator is [Os(nXbpy)2(im)Cl] + [Cir, the enzyme is glucose oxidase, the buffer is MES
buffer, the film former is polyvinyl alcohol, and the surfactant is a nonionic surfactant

23. An electrochemical sensor useful for measuring the concentration of an analyte
5 in a fluid sample, comprising:

(a) a first insulating substrate;

(b) working and counter electrodes, having first and second ends and a middle
portion, affixed to the first insulating substrate in a predetennined pattern, the
pattern being defined by a combination of photolithography and chemical

10 etching;

(d) a reagent disposed on at least the first end of the working electrode, the reagent
being specifically reactive with the analyte in the fluid sample to produce an

1 5 electrochemically-measurable signal which can be correlated to the concentration

of the analyte in the fluid sample.

24. The electrochemical sensor of claim 23, wherein the electrically conducting
material of me working electrode element is a noble metal.

25. The electrochemical sensor of claim 24, wherein the electrically conducting
20 material of the counter electrode element is a noble metal.

26. The electrochemical sensor of claim 25, further comprising:

(d) a power source in electrical connection with the second ends of the working and
counter electrodes; and

(e) a meter in electrical connection with the second ends of the working and counter
25 electrodes and capable of measuring current

27. The electrochemical sensor of claim 26, wherein the reagent comprises a
mediator and an enzyme.

WO 95/22597 PCT/US95/02157

28. The electrochemical sensor of claim 27, wherein the reagent further comprises a
buffer, a film former, and a surfactant

29. The electrochemical sensor of claim 27, wherein the mediator is
[Os(IIXbpy)2(im)CI] + [Cl]' and the enzyme is glucose oxidase.

5 30. The electrochemical sensor of claim 28, wherein the analyte is glucose, the

mediator is [OsCnXbpy^imX^l^tCl]", the enzyme is glucose oxidase, the buffer is MES
buffer, the film former is polyvinyl alcohol, and the surfactant is a nonionic surfactant.

3 1 . A method for manufacturing an electrode element for use in an electrochemical
sensor, comprising:

10 (a) affixing an electrically conducting material to a first insulating substrate;

(b) coating the electrically conducting material with a second insulating substrate,
the second insulating substrate being insoluble in a developer solution after
exposure to ultraviolet light;

(c) exposing the second insulating substrate to ultraviolet light through a photomask,
1 5 such that a portion of the second insulating substrate is rendered insoluble to the

developer solution; and

(d) exposing the second insulating substrate to the developer solution, thereby
removing the soluble portion of the second insulating substrate and exposing first
and second cutout portions, the first cutout portion acting as an electrode area

20 and the second cutout portion acting as a contact pad between the electrically

conducting material and a meter and a power source.

32. The method of claim 31, wherein the electrically conducting material is a noble
metal or carbon.

33. The method of claim 32, wherein the second insulating substrate is a solder

25 resist

34. The method of claim 33, wherein the electrically conducting material is
deposited on the first insulating substrate by vacuum sputtering or evaporative deposition.

WO 95/22597 PCIYUS95/02157

35. The method of claim 33, wherein the electrically conducting material is
deposited on the first insulating substrate by electroless plating or electroplating.

36. The method of claim 31, wherein the electrically conducting material is a noble
metal or carbon and is affixed to a thin support material before being affixed to the first

5 insulating substrate.

37. The method of claim 36, wherein the thin support material is polyimide,
polyester, PET, or polycarbonate.

38. The method of claim 31, wherein the electrically conducting material is silver
and the electrode area is coated with silver chloride.

1 0 39. The method of claim 38, wherein the second insulating substrate is a solder

resist.

40. The method of claim 39, wherein the electrically conducting material is
deposited on the first insulating substrate by vacuum sputtering or evaporative deposition.

41. The method of claim 39, wherein the electrically conducting material is
1 5 deposited on the first insulating substrate by electroless plating or electroplating.

42. The method of claim 31, wherein the electrically conducting material is silver
and is affixed to a thin support material before being affixed to the first insulating substrate.

43. The method of claim 42, wherein the thin support material is polyimide,
polyester, PET, or polycarbonate.

20 44. A method for manufacturing a counter electrode element for use in an

electrochemical sensor, comprising:

(a) affixing an electrically conducting material to a first substrate, the first substrate
including a copper layer and a fiberglass layer, the copper layer being disposed
between the electrically conducting material and the fiberglass layer; and
25 (b) attaching a spacer to the electrically conducting material, the spacer having a

first cutout portion which defines the electrode area and a second cutout portion
which allows contact between the electrically conducting material and a meter
and a power source.

WO 95/22597

PCT/US95/02157

45. The method of claim 44, wherein the electrically conducting material is affixed
to the copper layer of the first substrate by screen printing.

46. The method of claim 45, wherein the electrically conducting material is a noble
metal or carbon.

5 47. The method of claim 45, wherein the electrically conducting material is silver

and is coated with silver chloride, thereby forming a reference electrode.

48. The method of claim 44, wherein the electrically conducting material is affixed
to the copper layer of the first substrate by laminating.

49. A method for manufacturing an electrode element for use in an electrochemical
1 0 sensor, comprising:

(a) affixing a first electrically conducting material to a first insulating substrate;

(b) affixing a second electrically conducting material to the first electrically
conducting material; and

1 5 having a first cutout portion which defines the electrode area and a second cutout

portion which allows contact between the electrically conducting material and a
meter and a power source.

50. The method of claim 49, wherein the first electrically conducting material is
palladium.

20 51. The method of claim 50, wherein the first electrically conducting material is

affixed to a thin support material, which is affixed to the first insulating substrate.

52. The method of claim 51, wherein the second electrically conducting material is

silver.

53. The method of claim 52, wherein the first electrically conducting material is
25 deposited on the thin support material by vacuum sputtering or evaporative deposition.

54. The method of claim 53, wherein the first electrically conducting material is
deposited on the thin support substrate by electroless plating or electroplating.

55. The method of claim 54, further comprising:

WO 95/22597

PCT/US95/02157

(e) dispensing FeCl 3 onto the silver exposed by the first cutout portion of the spacer,
thereby forming a layer of silver chloride.

56. A method for manufacturing electrode elements for use in an electrochemical
sensor, comprising:

5 (a) affixing an electrically conducting material to a thin support material;

(b) affixing die thin support material to a first insulating substrate;

(d) exposing the photoactive etch resist to ultraviolet light through a photomask,
such that a portion of the photoactive etch resist is rendered insoluble to a

1 0 developer solution by exposure to the ultraviolet light;

(e) exposing the photoactive etch resist, after ultraviolet light exposure, to the
developer solution, thereby removing portions of the photoactive etch resist to
expose the electrically conducting material;

(f) exposing the photoactive etch resist and the electrically conducting material, after
1 5 exposure to the developer solution, to a chemical etchant, thereby removing

portions of die electrically conducting material not covered by the photoactive
etch resist; and

(g) removing the remaining photoactive etch resist, thereby exposing an electrode
pattern of electrically conducting material on the first insulating substrate.

20 57. The method of claim 56, wherein the electrode pattern of electrically conducting

material includes a working electrode and a counter electrode.

58. The method of claim 57, wherein die electrically conducting material is a noble

metal.

59. The method of claim 58, wherein the thin support material is polyimide,
25 polyester, PET, or polycarbonate.

60. The method of claim 59, wherein the electrically conducting material is
deposited on the thin support material by vacuum sputtering or evaporative deposition.

WO 95/22597 PCT/US95/02157

61 . The method of claim 59, wherein the electrically conducting material is
deposited on the thin support material by electroless plating or electroplating.

62. The method of claim 57, further comprising:

(g) converting the counter electrode to a silver/silver chloride reference electrode by •
5 depositing silver on the surface of the counter electrode and treating the silver

withFeCl 3 .

WO 95/22597

PCI7US95/02157

Fig. 2

SUBSTITUTE SHEET (RULE 26)

WO 95/22597

PCT/US95/02157

SUBSTITUTE SHEET (RULE 26)

WO 95/22597

4/5

PCT/US95/02157

SUBSTITUTE SHEET(RULE 26)

WO 95/22597

5/5

PCT/DS95/02157

SUBSTITUTE SHEET (RULE 26)

INTERNATIONAL SEARCH REPORT

Internationa] application No.
PCTAJS95/02157

A. CLASSIFICATION OF SUBJECT MATTER
IPC(6) :C12M 1/40; C25B 1 1/00

USCL :435/288; 427/125
According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols) ~~" "~ -—*-—*■
U.S. : Please See Extra Sheet.

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US, A, 4,894,137 (TAKIZAWA ET AL.) 16 January 1990,
see entire document.

23-26, 31, 32

27-30, 33-43,
56-62

X
Y

US, A, 5.108,819 (HELLER ET AL.) 28 April 1992, see entire
document.

31-34, 36, 37,
49-51

23-30, 35, 38-
43, 52-62

fx] Further documents are listed in the continuation of Box C. [ j See patent family annex.

Special caieforics of cited

deanKBtdefinittg the acncral state of the en which m tv* considered
to be of particular relevance

enrBcr document pobBshcd en or after the international fflinf dale

docycal which may throw doubts oo priority cbura(>) or which m
cited to establish the publication date of

doctnna

•X*

rin| to an oral disclosure, use, exhibition or other

later oocwnenl published after the mtcrnatianal film* date or priority
date and not in conflict with the appHcatioobtitcr^ toundcrsta«Jtbe
priocjpk or theory undeiiymj the invention

document of particular re l evanc e ; the churned mvemioo cannot be
considered novel or cannot be considered to involve sn mventive step
when the document is taken akme

document of particular relevance; the churned invention cannot be
considered to involve ma inventive step when the document is

djoewnent pnbBahed prior to jhefacw8tfonal|ihB^dj^b^ii|sj|gr^inj

bebf obvious to a person skilled in the art
r of the sane patent family

Date of the actual completion of the mternational search
17 MAY 1995

Date of mailing of the mternational search report

07 JUN 1995

Name and mailing address of the ISA/US
Commissioner of Patents and Trademarks
BoxPCT

Washington. D.C. 20231
Facsimile No. (703)305-3230

WILLIAM H. BE1SNER (J ^^^V
Telephone No. (703) 308-0196

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US95/02157

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US, A, 5,288,636 (POLLMANN ET AL.) 22 February 1994, see
entire document.

29, 30, 36, 37,
42, 43, 53-62

v
A

Jf, A, 1 IL (MA lou orLl 1 A rxJiU. UNJJ. Jvlv) lo UCtOber

1979, see entire document.

44, 49

45-48, 50-55

US, A, 4,929,426 (BODAI ET AL,) 29 May 1990, see entire
document.

44-48

49-55

DE, B, 2,122,608 (RICHLY ET AL.) 20 April 1978, see entire
document.

1-62

JP, A, 63-300,954 (SHIMADZU SEISAKUSHO KK) 08
December 1988, see entire document.

1-62

JP, A, 62-32,351 (MORHZUMI) 12 February 1987, see entire
document.

1-62

US, A, 4,882,839 (OKADA) 28 November 1989, see entire
document.

1-62

Form PCT/1S A/210 (continuation of second sheet) (July 1992)*

INTERNATIONAL SEARCH REPORT

international application No.
PCT/US95/02157

B. FIELDS SEARCHED
Minimum documentation searched
Classification System: U.S.

435/28$, 291, 817; 422/82.01, 82.02; 204/153.1, 153.12, 403, 280, 290R, 291; 427/2.12, 2.13, 10, 58, 123-125, 508
510, 511, 551, 553, 558, 58, 96, 115

Form PCT/ISA/210 (extra sheetXJuly 1992>*

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